Effect of hypoxia on GLP-1 secretion – an in vitro study using enteroendocrine STC-1 -cells as a model

Glucagon-like peptide (GLP)-1 is a hormone released by enteroendocrine L-cells after food ingestion. L-cells express various receptors for nutrient sensing including G protein-coupled receptors (GPRs). Intestinal epithelial cells near the lumen have a lower O2 tension than at the base of the crypts, which leads to hypoxia in L-cells. We hypothesized that hypoxia affects nutrient-stimulated GLP-1 secretion from the enteroendocrine cell line STC-1, the most commonly used model. In this study, we investigated the effect of hypoxia (1% O2) on alpha-linolenic acid (αLA) stimulated GLP-1 secretion and their receptor expressions. STC-1 cells were incubated for 12 h under hypoxia (1% O2) and treated with αLA to stimulate GLP-1 secretion. 12 h of hypoxia did not change basal GLP-1 secretion, but significantly reduced nutrient (αLA) stimulated GLP-1 secretion. In normoxia, αLA (12.5 μM) significantly stimulated (~ 5 times) GLP-1 secretion compared to control, but under hypoxia, GLP-1 secretion was reduced by 45% compared to normoxia. αLA upregulated GPR120, also termed free fatty acid receptor 4 (FFAR4), expressions under normoxia as well as hypoxia. Hypoxia downregulated GPR120 and GPR40 expression by 50% and 60%, respectively, compared to normoxia. These findings demonstrate that hypoxia does not affect the basal GLP-1 secretion but decreases nutrient-stimulated GLP-1 secretion. The decrease in nutrient-stimulated GLP-1 secretion was due to decreased GPR120 and GPR40 receptors expression. Changes in the gut environment and inflammation might contribute to the hypoxia of the epithelial and L-cells. Graphical Abstract

Oxygen (O 2) is a key factor affecting GLP-1 synthesis and release [55].The epithelial cells near the lumen have a much lower O 2 tension than at the base of the crypts with a steep oxygen gradient, which is critical for intestinal homeostasis [46].The O 2 tension in the villus falls significantly from 14-17 mmHg in the interdigestive phase to 4-7 mmHg postprandial [17].Furthermore, O 2 levels drop from 10-7.5% in the stomach to 1.5-0.5% at the colon-rectal junction [58].These anatomical characteristics provide a sharp oxygen gradient across the tissue, making intestinal cells hypoxic even under physiological conditions, known as 'physiological hypoxia'.As a consequence, the colonic microbiome is primarily composed of anaerobic bacteria, predominantly Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia [41].Anaerobes further contribute to the intestine's hypoxic environment by utilizing part of the O 2 from the bloodstream [8].
Hypoxia induces the stabilization of the transcription factors hypoxia-inducible factor-1α (HIF-1α) and HIF-2α which is influenced by O 2 -regulated proteins such as prolyl-hydroxylases (PHDs) and factor-inhibiting HIF (FIH) [21].The HIF-α subunit has three isoforms in vertebrate species: HIF-1α, HIF-2α, and HIF-3α.These proteins enter the nucleus and form complexes with the constitutively produced β subunit, regulating gene expression of several fundamental cellular processes such as metabolism and autophagy.

STC-1 cell culture
The STC-1 cell line was cultured in T75 flasks in DMEM supplemented with 15% horse serum, 2.5% fetal bovine serum (FBS), 1% L-glutamine, and penicillin-streptomycin as previously described [40].Cells were maintained in the above media at 37˚C, 5% CO 2 until 85-90% confluency.Passage numbers 14-21 were used for the experiment.The mouse STC-1 cell line is the most commonly used GLP-1 secreting cell line and serves as a model cell line in the investigations of GLP-1 [26,29,40,48].In addition, other cell lines like mouse GLUTag cells secrete significantly less GLP-1 [29].Human cell lines secreting GLP-1 are the colon cancer cell line Caco-2 and the NCI-H716 cell line from a 33-yr-old Caucasian male with poorly differentiated adenocarcinoma of the colon [42,50].Cell line models of L-cells differ from each other and from the natural L-cells [36].However, there is abundant GLP-1 in the distal intestine in different species like mice, rats, pigs and humans responding to nutrient stimulation [30].

GLP-1 secretion in STC-1 cells under normoxia and hypoxia
STC-1 cells were seeded at 2.0 × 10 6 cells/well with 2 ml media in 6-well cell culture plates.Cells were counted by an automated cell counter (LUNA-II™, Logos Biosystems, Inc., Villeneuve-d´Ascq, France).On the day of experiments, cells were washed and incubated with DMEM for 12 h in normoxia (18% O 2 , 5% CO 2 ) and hypoxia (1% O 2 , 5% CO 2 ) using the hypoxic chamber, Sci-Tive-N/ Ruskinn, Baker, Sanford, ME.After 12 h of incubation, the cells were washed twice with Krebs-Ringer Bicarbonate Buffer (KREBS; 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO 3 , 1.25 mM CaCl 2 , 1.2 mM MgSO 4 , and 1.2 mM KH 2 PO 4 ) pH 7.4.Hypoxia was maintained throughout the experiment by keeping the cells in a hypoxia chamber and all the treatments including washing were performed inside the chamber.Following washing cells were acclimatized with KREBS buffer for 1 h.After 1 h of acclimatization, the buffer was aspirated, and cells were treated in triplicates with different treatments along with DPP4 inhibitor in KREBS buffer for 1 h at 37 • C: αLA (12.5 μM) as shown in Fig. 1 [48].KREBS buffer with ethanol (0.1%) and DPP4 inhibitor (0.25%) was used as a control.After 1 h of treatment, the supernatant was collected, centrifuged at 13,000 g for 15 min at 4 • C and stored at -70 • C. Cells were washed with ice-cold PBS for protein analysis.Active GLP-1 was measured by an active GLP-1 ELISA kit as per manufacturer instructions [29] (n = number of individual experiments performed in triplicates).

Immunoblotting for protein analysis
Cells were scrapped from a 6-well plate using a cell scrapper and ice-cold RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), containing protease inhibitor cocktail (Sigma, St. Luis, MO).The suspension was passed using a 27G needle and centrifuged at 13,000 g for 20 min at 4˚C.Total protein concentrations were determined by the Bradford reagent (Bio-Rad Laboratories Inc., Hercules, CA).Equal amounts of protein (12.5 μg) were separated by 8% SDS-PAGE gel and transferred to a PVDF membrane.The membrane was blocked using 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and incubated with primary HIF-1α monoclonal antibody (1:1000), GPR120 polyclonal antibody (1:1000), GPR40 polyclonal antibody (1:1000) and loading control protein GAPDH (1:1000) overnight at 4˚C.After overnight incubation, the membrane was incubated with a secondary antibody (1:10,000 Goat anti-rabbit IgG horseradish peroxidase-conjugated anti-rabbit IgG) for 1 h at room temperature.Chemiluminescence for proteins was detected using SuperSignal™ West Femto Maximum Sensitivity Substrate according to the manufacturer´s instructions.Blots were visualized with an Odyssey Fc imaging system (LI-COR Biosciences, Ltd, Cambridge, UK).

Statistical analysis
One-way analysis of variance (ANOVA) was used to analyze statistical significance between the groups using GraphPad Prism, version 7 (GraphPad Software, Inc., La Jolla, CA).Dunnett's multiple comparison test was used to analyze the difference between the treatment groups.The values are represented as mean ± standard errors of the mean (SEM) and differences were considered statistically significant when p < 0.05.

Discussion
Our study demonstrated that nutrient αLA significantly stimulated GLP-1 secretion from enteroendocrine STC-1 cells under normoxia but not under hypoxic conditions.However, no significant change in GLP-1 secretion was found under basal conditions between hypoxia and normoxia.αLA was used as a positive control to stimulate GLP-1 secretion in STC-1 cells as previously described [48].Our results are consistent with previous findings under normoxia on GLP-1 secretion with fatty acids in STC-1 cells [29,48].Consistent with our findings on hypoxia, Kondrashina et al. showed that casein-stimulated GLP-1 secretion significantly decreased under hypoxia (7.5% O 2 ) in STC-1 cells [34].In addition, hypoxia (1% O 2 ) decreased forskolin-stimulated GLP-1 secretion from enteroendocrine GLUTag cells [32].The author suggested that the decrease in GLP-1 secretion could be due to reduced GLP-1 synthesis [32].6-168 h of hypoxia (simulated altitude of 7620 m) in rats decreased plasma CCK and GLP-1 [15].In contrast, it has been shown that 64 cycles of intermittent hypoxia for 5 min (1% O 2 ) and 10 min normoxia (21% O 2 ) increased mRNA expression of PYY and GLP-1 in STC-1 cells [45].Chronic hypoxia (14.4-14.7%O 2 ) for four weeks, lowered body weight, and improved glucose, and lipid metabolism in HFD-induced obese mice [54].In humans, normobaric hypoxia (fraction of inspired oxygen: FiO 2 :0.12 ~ 5000 m) for 10 h reduced appetite and hunger compared to normobaric normoxia (FiO 2 :0.21), but gut peptides were unfortunately not measured [4].17 h Fig. 2 Active GLP-1 secretion under normoxia and hypoxia in STC-1 cells.12 h hypoxia did not significantly affect basal GLP-1 secretion compared to normoxia control.αLA significantly stimulated GLP-1 secretion under normoxia (~ 7 folds), but no significant change under hypoxia compared to buffer (control).In addition, αLA-stimulated GLP1 secretion under hypoxia was significantly lower compared to normoxia αLA.n = 5 (n = number of individual experiments in triplicates).The value represents the mean ± standard error of the mean (SEM) and differences were considered statistically significant when ***p < 0.001 and ****p < 0.0001 hypoxic exposure (12.5% inspired O 2 , simulating approximately ~ 4100 m) in humans increased plasma leptin and only a small increase in postprandial GLP-1 levels after 40 min [47].In humans, reduced energy intake, plasma acylated ghrelin concentrations, and PYY concentrations were reported at a simulated altitude of 4000 m [56].Matu et al. reported lower appetite and post-exercise acetylated ghrelin area under the curve at an altitude of 4300 m, but no change in GLP-1 secretions [37].A reason might be that no DPP4 was added for the measurement of GLP-1.
Several peptides including GLP-1 are secreted from the intestinal tract, which has a distinct oxygen gradient.A vertical oxygen gradient has been found in the more distal colonic parts of the gastrointestinal (GI) tract.The epithelial cells lining of intestinal mucosa reside in a relatively low pO 2 environment [10].The pO 2 of the colonic muscle wall is 42-71 mmHg (7-10%), vascularized submucosa 42 mmHg (6%), crypt-lumen interface 5-10 mmHg, ascending colon 11 mmHg (2%) and sigmoid colon 3 mmHg (0.4%) [25].During low-oxygen conditions, cells adapt to hypoxic stress by increasing the HIFs expression, which regulates metabolic processes and energy metabolism [43].Hence in our study, we incubated STC-1 cells for 12 h (1% O 2 , 5% CO 2 ) to induce and stabilize HIF-1α.HIF-1α protein expressions clearly state that hypoxia was maintained throughout the experiment (Fig. 3).We observed faint HIF-1α protein bands under normoxia, which has also been reported in other studies [18,19,49].Acute infection and inflammation cause significant changes in tissue metabolism, resulting in severe tissue hypoxia [22].A study on murine dendritic cells subjected to hypoxia (2% oxygen for 24 h) demonstrated an elevation in transcript levels of Toll-like receptors TLR2 and TLR6 [35].In addition, hypoxic environment affects the intestinal microbiome.Following a high-fat diet (HFD) through meat consumption, both humans and animals showed a higher abundance of Firmicutes and a decrease in Bacteroidetes.Changes in the gut microbiota populations activate the Toll-like receptor (TLR) signaling pathway, resulting in increased intestinal permeability to endotoxins [12].In mice colon, Bacteroidetes were reported about 3 times higher in abundance during hypoxia (simulated altitude of 5500 m for 24 h) [53].In mice, the ratio of total aerobic to anaerobic bacteria changed from 1:2.79 to 1:7.34 under exposure to a highaltitude environment for 30 days and the total number of anaerobes increased about 105 times higher [1].The Bacteroidetes abundance increased from days 1 to 14 in mice during environmental hypoxia with HIF-1ß deficiency in their bone marrow cells [23].These disturbances in gut microbiota might cause a reduction in GLP-1 secretion.Colonocytes use butyrate produced by the bacteria as an energy substrate and maintain the anaerobic environment in the lumen [11].In Caco-2 cells, butyrate increased O 2 consumption and stabilized HIF-1α, leading to lower barrier permeability [31].In human NCI-H716 cells, butyrate stimulated dose-dependently and biphasic GLP-1 secretion [57].Yet, butyrate promotes epithelial barrier function by depleting oxygen levels near the epithelium and stabilizing HIF [3], stimulating mucin production [24].The microbiome generates biofilms, which further contributes to the decreased oxygen gradient in the colon [5,51].Oxygen concentrations are high at the liquid surface and low in the deeper parts of the biofilm.In addition, the oxygen diffusion rate (about 60% of the rate observed in water) is lower in biofilms [33] which also contributes to the hypoxic environment in the colon.
Intestinal HIF affects GLP-1 secretion via the lipid sensor G-protein-coupled receptor enriched in L-cells.The enteroendocrine cells sense fatty acids (FAs) via GPR120 and GPR40 [52].Thus, we investigated GPR120 and GPR40 protein expressions.We found that hypoxia (increased HIF-1α) decreased αLA stimulated GPR120 and GPR40 protein expression (Fig. 3).The decreased GLP-1 secretions could be due to receptor internalization.In addition, it has been shown that HIF-2α increased GPR40 in L-cells and potentiates fatty acid-induced GLP-1 secretion via extracellular signal-regulated kinase (ERK) [38].In our study, under hypoxia, GPR120 and GPR40 were downregulated, which led to decreased GLP-1 secretion.Previously it has been shown that dual GPR120 and GPR40 agonist (DFL23916) delays receptor internalization and induce GLP-1 secretion in vitro and in vivo [6].Internalization of receptors under hypoxia has been described in the brain before.Hypoxiainduced desensitization and internalization of adenosine A1 receptors in the rat hippocampus and internalization of Kappa (κ) opioid receptors [9].

Conclusions
Hypoxia did not affect the basal GLP-1 secretion but decreased nutrient-stimulated GLP-1 secretion in enteroendocrine cells.In addition, hypoxia decreased the expression of long-chain fatty acid receptors GPR120 and GPR40.The decrease in nutrient-stimulated GLP-1 secretion could be due to downregulations of GPR120 and GPR40 receptors.Changes in the gut environment and inflammation might contribute to the hypoxia of the epithelial and L-cells.

Fig. 3
Fig. 3 Representative western blots of HIF-1α in STC-1 cells.In hypoxic conditions, all the blots showed intense HIF-1α protein bands at ~ 120 kDa and only faint bands under normoxia.A) 12 wells of 2 experiments in triplicates.B) 4 wells from one experiment in